Known methods for measuring the activity of lipase include olive oil method (1) and colorimetric methods (2) to (4) as explained below.
(1) The first method involves reacting lipase with emulsified olive oil or synthetic triglyceride as a substrate and neutralizing and titrating the fatty acid formed by hydrolysis, or extracting the fatty acid, coloring sodium diethylthiocarbamate, and then colorimetrically measuring the color. Alternatively, this method may involve reacting lipase with an emulsified substrate and measuring the reduction of turbidity by the hydrolysis. Typical examples are described in J. H. Roe, Analytical Biochemistry, 6, 451 (1963).
(2) The second method is a colorimetric method of reacting lipase with an ester of a phenol such as .alpha.-naphthol, phenol, p-nitrophenol, etc., and a long chain fatty acid as a substrate, coloring a phenol formed by hydrolysis, and measuring the extent of the hydrolysis. Typical examples are described in F. Whitaker, Clinica Chimica Acta 44, 133 (1973).
(3) The third method involves reacting an S-acyl compound such as 3-mercapto-1,2-propanediol tricarboxylic acid ester, etc. with lipase to hydrolyze the S-acyl compound, reacting the thiol formed with 5,5'-dithiobis(2-nitrobenzoic acid) to form a yellow dye, 3-carboxy-4-nitrobenzenethiolate, and measuring the color by colorimetry. These methods are described in, for example, Japanese Patent Application (OPI) Nos. 33694/76, 5309/75, 151594/75 and 159793/75 (the term "OPI" as used herein refers to a "published unexamined Japanese patent applicaton").
(4) The fourth method involves hydrolyzing a higher fatty acid ester of naphtholic orange azo dye by the action of lipase and measuring the orange dye formed by colimetry. Such methods are described in Japanese Patent Application (OPI) No. 46758/79 corresponding to U.S. Pat. No. 4,188,320.
In method (1), it is difficult to prepare a stable and uniform substrate emulsion and there are problems in operability and reproducibility. In methods (2) and (3), since the substrate is insoluble in water, the enzymatic reaction must be performed in the form of emulsion or must be performed in the presence of an organic solvent such as an alcohol. Furthermore, deproteinization must be performed after the reaction, which makes the operation complicated. These difficulties may be overcome in method (4) but since the dye measured by colorimetry in method (4) is orange, the colorimetric method is distributed by colored matters such as dyes in blood. Therefore, it would be more desirable to develop a method which would release a dye having a light absorption zone at a longer wavelength region. Also, further improvement has been desired regarding the spectral extinction coefficient of the released dye having an influence on the measurement sensitivity.